Electron beam lithography of poly(glycidol) nanogels for immobilization of a three-enzyme cascade

Surface immobilization of multiple enzymes plays an increasingly important role in biosensor development. Traditional nanofabrication methods require multiple fabrication steps and complex syntheses which often result in limited enzyme density, modest spatial control and decreased enzyme activity over time and repeated uses, particularly when extensive synthetic modifications are applied. We employed electron beam lithography to crosslink poly(glycidol) with pre-loaded enzyme nanogels, which contain crosslinked poly(glycidol), on a silicon surface to enhance the stability and robustness of synergistic enzymes. As proof-of-concept, we immobilized three enzymes β-galactosidase, glucose oxidase, and horseradish peroxidase with and without nanogel pre-entrapment, and bioactivity was investigated via colorimetric determination with lactose substrate and o-phenylenediamine indicator. The resulting enzyme nanogel device exhibited 71% retention of bioactivity after 30 days and 5-fold higher chromogenic output compared with 32% from free enzyme devices.